The first approach will examine further the segregation of acetylcholine receptors (AChR) induced by cholesterol enrichment in chick myocytes. In the chick myocyte, the cholesterol enrichment induced two ACHR channel conductances (51 & 39 pS). A working hypothesis is that cholesterol induces lateral phase separations that alter the local concentration and degree of interaction of cholesterol with the AChR. The local cholesterol environment exerts an allosteric effect on the ACHR channel properties. The effect of temperature on the heterogeneous distribution of AChRs induced by cholesterol will be examined. Cholesterol depletion studies will be performed to examine AChR channel function in a membrane with reduced cholesterol levels. General anesthetics will be used to probe the hydrophobic environment of the 56 and 39 pS AChR channel in the cholesterol enriched myocyte. The second approach is to extend the site-directed mutagenesis analysis of the lipid exposed residues of the Torpedo californica AChR to the M3 transmembrane segment. The M3 has a similar degree of contact with the lipid when compared to the M4. Particular attention will be given to M3 positions on the four subunits (alpha, a, beta, b, gamma, g, and delta, d) equivalent to the previous M4 mutations that produced significant effects on channel gating. Phenylalaline and tryptophan substitutions are the first to be introduced since in previous M4 substitutions, these side chains have been shown to be especially effective in altering ion channel gating. Mutants will be examined in Xenopus laevis oocytes using voltage-clamp and patch clamp electrophysiology. The third approach to be used is to incorporate unnatural amino acids into lipid exposed positions using the nonsense suppressor technique to define the structural and functional linkage between lipid exposed residues and the AChR channel gating (specific aim 3). A working hypothesis is that indole dipole moments are specially effective in producing such effects on the AChR channel gating. Phenylalanine and tryptophan analogs will be introduced at sensitive positions of the M3 and M4 transmembrane segments to define the structural linkage between lipid exposed residues and AChR channel function.